New Approaches Toward Recognition of Nucleic Acid Triple Helices DEV P. ARYA* Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, South Carolina 29634, United States RECEIVED ON AUGUST 12, 2010
CONSPECTUS
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DNA duplex can be recognized sequence-specifically in the major groove by an oligodeoxynucleotide (ODN). The resulting structure is a DNA triple helix, or triplex. The scientific community has invested significant research capital in the study of DNA triplexes because of their robust potential for providing new applications, including molecular biology tools and therapeutic agents. The triplex structures have inherent instabilities, however, and the recognition of DNA triplexes by small molecules has been attempted as a means of strengthening the three-stranded complex. Over the decades, the majority of work in the field has focused on heterocycles that intercalate between the triplex bases. In this Account, we present an alternate approach to recognition and stabilization of DNA triplexes. We show that groove recognition of nucleic acid triple helices can be achieved with aminosugars. Among these aminosugars, neomycin is the most effective aminoglycoside (groove binder) for stabilizing a DNA triple helix. It stabilizes both the TAT triplex and mixed-base DNA triplexes better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (TAT and mixed base) without any effect on the DNA duplex. The selectivity of neomycin likely originates from its potential and shape complementarity to the triplex Watson-Hoogsteen groove, making it the first molecule that selectively recognizes a triplex groove over a duplex groove. The groove recognition of aminoglycosides is not limited to DNA triplexes, but also extends to RNA and hybrid triple helical structures. Intercalator-neomycin conjugates are shown to simultaneously probe the base stacking and groove surface in the DNA triplex. Calorimetric and spectrosocopic studies allow the quantification of the effect of surface area of the intercalating moiety on binding to the triplex. These studies outline a novel approach to the recognition of DNA triplexes that incorporates the use of noncompeting binding sites. These principles of dual recognition should be applicable to the design of ligands that can bind any given nucleic acid target with nanomolar affinities and with high selectivity.
1. Introduction
molecules such as oligodeoxynucleotides (ODNs, major
Out of the earth shall come thy salvation. Selman Waksman
groove binders).1,2 Major groove recognition of the duplex DNA by ODNs results in a triple helix in a parallel or antiparallel form (Figure 1). Triple helix formation was first
Duplex DNA can be sequence-specifically recognized by small molecules such as minor groove binders and larger 134
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reported in 1957 by Felsenfeld and co-workers.3,4 The elucidation of the DNA duplex structure at the same time Published on the Web 11/12/2010 www.pubs.acs.org/accounts 10.1021/ar100113q & 2010 American Chemical Society
Recognition of Nucleic Acid Triple Helices Arya
FIGURE 1. Base interactions in parallel (pyrimidine motif, top) and antiparallel (purine motif, bottom) triple helices. These are defined with respect to the orientation of the TFO and homopurine Watson-Crick (W-C) strand.
kept the limelight away from the three-stranded DNA strucle ne, tures until the 1980s. Eventually, work by Dervan, He and others showed the broad potential of triplex-forming ODNs in targeting duplex and single-stranded DNA and in inhibiting sequence-specific DNA-protein interactions.5-9 DNA triplex formation has continued to garner much interest in the scientific community because of the possible applications in developing new molecular biology tools as well as therapeutic agents.10-13 Specific inhibition of transcription has been induced via triplex formation at poly(purine/pyrimidine) sites in promoter sequences. These and numerous other findings that support the feasibility of an antigene approach for therapeutically regulating specific gene expression have been discussed in a number of reports.14-16 A recent report has postulated that DNA triplexes may even be present at the active site of the DNA polymerases.17
Several intercalators as well as various DNA minor groove ligands have previously been shown to bind to DNA triple helices.18,19,19-21 Intercalators usually stabilize to a greater extent triple helices containing TAT triplets, whereas minor groove binders usually destabilize triplexes.22 At the beginning of this millennium, when we began our work on recognition of nucleic acids, one surprising fact stared at us: While there were a number of intercalators that stabilized DNA duplex and triplex structures (selectively and nonselectively), there were no examples of groove-binding ligands that selectively recognized the triple helices (spermine,23 with its flexible amines as the possible weak exception). This was in stark contrast to the numerous sequence-specific groove binders known to bind in the minor groove of duplex DNA.2,24,25 Time and again, one has had to look to natural products to deal with such problems in recognition of biomolecules. Examples of such Vol. 44, No. 2
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FIGURE 2. Structures of some aminoglycoside antibiotics. Ring numbering scheme is shown for neomycin.
natural products include crescent shaped netropsin and distamycin26-28 which were key leads in the design principles that are used to target the DNA minor groove today. In such a quest for ligands for triple-helix-specific stabilization, we decided to investigate aminoglycoside antibiotics, the antibacterial scaffolds first discovered by Waksman in 1944 for activity against tuberculosis (Figure 2).29-32 In this Account, I will present neomycin as one of the first examples that bridge the gap in molecular recognition of triplex structures and increase our understanding of the recognition principle(s) involved in selective targeting of nucleic acid triplex grooves.
2. Thermal Denaturation Studies and Aminoglycoside Structure-Activity Relationships Thermal denaturation studies of triplexes formed from
FIGURE 3. Plots of variation of triplex melting (Tm3f2) and duplex melting (Tm2f1) of poly(dA) 3 2poly(dT) as a function of increasing neomycin concentration {rdb = drug(neomycin)/base triplet ratio}. Conditions: 10 mM sodium cacodylate, 150 mM KCl, pH 6.8, [poly(dA) 3 2poly(dT)] = 15 μM/base triplet. Reprinted with permission from ref 33. Copyright 2010 Elsevier.
ODNs as well as polynucleotides were carried out in the presence of aminoglycosides, using UV spectroscopy at 260/280 nm. These studies showed the remarkable effectiveness of neomycin in stabilizing the triplex without affecting the duplex Tm.33 In the thermal denaturation analysis of poly(dA) 3 2poly(dT) bound to neomycin, plots of absorbance at 260 and 284 nm (A260, A284) versus temperature exhibit two distinct inflections {Tm3f2 (triplex melting point) = 34 °C and Tm2f1 136
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(duplex melting point) = 71 °C, rdb = 0.15 (ratio of drug(neomycin)/base triplet)}. Triplex stabilization was found to be dependent on neomycin concentration. Figure 3 shows that, by increasing the molar ratios of neomycin from 0 to 25 μM, rdb = 0-1.67, the triplex melting point is increased by close to 25 °C, whereas the duplex is virtually unaffected. Nature has provided us antibiotics to allow for a simplified version of aminoglycoside-triplex structure-activity
Recognition of Nucleic Acid Triple Helices Arya
FIGURE 4. Effect of aminoglycoside antibiotics on the melting of poly(dA) 3 2poly(dT) triplex (rdb = 1.67). Solution conditions: 150 mM KCl, 10 mM sodium cacodylate, pH 6.8, [poly(dA) 3 2poly(dT)] = 15 μM/base triplet. Number of amines in each antibiotic is shown in parentheses. Reprinted with permission from ref 34. Copyright 2001 American Chemical Society.
TABLE 1. 35 Fluorescence Derived AC50 Values for Aminoglycoside Binding to 50 -dA12-x-dT12-x-dT12-30 Triple Helix at pH 5.5 and 6.8 in 10 mM Sodium Cacodylate, 0.5 mM EDTA, and 150 mM KCla AC50 (μM) aminoglycoside
pH 6.8
pH 5.5
neomycin paromomycin ribostamycin
35.5 179.0 486.0
13.3 157.9 459.0
a
T = 10 °C, [DNA triplex] = 100 nM/strand, [thiazole orange] = 700 nM.
relationship (Figure 4). A few of these aminosugars can be viewed as simple variations of neomycin pharmacophore with functional group deletions. Most aminoglycosides with five or more amines are able to stabilize the triple helix (increasing ΔTm3f2, without significantly affecting the ΔTm2f1 values, Figure 4). The structural difference between paromomycin and neomycin is a positively charged amino group (present in neomycin), replacing a neutral hydroxyl (present in paromomycin). This leads to a difference of 16 °C in Tm3f2 values (rdb = 1.67) at 150 mM Kþ concentration. As also seen from Figure 4, neomycin is far more effective than paromomycin or ribostamycin in stabilizing triple helices.34 Since both paromomycin and ribostamycin can be derived by deletion of specific groups/rings in neomycin, these two aminoglycosides offer preliminary evidence for specific functional group (ring I amine) or ring (ring IV) participation in DNA triplex binding by neomycin. A fluorescent intercalator displacement (FID) assay was then used to determine the AC50 values for the three aminoglycosides with poly(dA) 3 2poly(dT) and an intramolecular 50 -dA12-x-dT12-x-dT12-30 (x = hexaethyleneglycol
linker) triplex. These studies have been recently published, and the results are summarized below.35 The AC50 values reported in Table 1 are the aminoglycoside concentrations required to displace 50% of thiazole orange from the triplex, as measured by a decrease in 50% of the fluorescence. At pH 6.8, the neomycin AC50 (AC50 = 35.5 μM) is 6-fold lower than that of paromomycin (AC50 = 179 μM) and approximately 14-fold lower than that of ribostamycin (AC50 = 486 μM). A similar trend, with neomycin (AC50 = 13 μM) < paromomycin (AC50 = 157 μM) < ribostamycin (AC50 = 459 μM) is observed at pH 5.5. Studies with the poly(dA) 3 2poly(dT) triplex showed the same trends in AC 50 values. When the FID experiments were carried out with poly(dA) 3 poly(dT) duplex, thiazole orange could not be displaced by up to 10 mM neomycin, suggesting that the affinity of the aminoglycoside is 2-3 orders of magnitude lower for the AT rich DNA duplexes, when compared to its affinity for the AT rich DNA triplex. These results suggested that (i) paromomycin is much weaker in stabilizing the triplex (amino group in ring I is a key element in DNA triplex recognition); (ii) ribostamycin and neamine are much weaker in stabilizing the triplex, and both have similar effect on triplex stabilization (ring IV amines are involved in recognition; and ring III likely provides the neomycin conformer necessary for successful binding).
3. Stabilization of DNA Triple Helix Poly(dA) 3 2poly(dT) by Other Ligands34 A comparison with DNA groove binders indicates that neomycin is much more active than the minor groove binders (Figure 5) in stabilizing the triplex. The minor groove binders, in general, are not triplex-specific (berenil, distamycin, Hoechst dyes), and some are known to even destabilize the triplex (berenil, distamycin) because of their preference for the DNA duplex.34 The minor groove binders such as Hoechst 33258 stabilize the DNA duplex much better than the DNA triplex, suggesting that the significant perturbations take place in the minor groove upon ODN binding to the duplex major groove. Binding studies suggest that neomycin, as opposed to minor groove DNA duplex binders, can preferentially bind the DNA triplex grooves over those present in the AT rich DNA duplex. Structural data identifying specific groove-drug interactions would further clarify which of the three triplex grooves are being targeted. The intercalating ligands, on the other hand, are equally or more effective in stabilizing the triple helix at low concentrations.34 At higher concentrations, the intercalating ligands Vol. 44, No. 2
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FIGURE 5. (a) Structures of groove binders (left) and intercalators (right), known to bind to DNA triplexes. (b) Effect of 10 μM (rdb = 0.66) groove binders on the DNA triplex melt {poly(dA) 3 2poly(dT)} (black) and the duplex melt {poly(dA) 3 poly(dT)} (boxed). Distamycin does not show a Tm3f2 transition (
